Dataset Information

Mu-seq: sequence-based mapping and identification of transposon induced mutations.

ABSTRACT: Mutations tagged by transposon insertions can be readily mapped and identified in organisms with sequenced genomes. Collections of such mutants allow a systematic analysis of gene function, and can be sequence-indexed to build invaluable resources. Here we present Mu-seq (Mutant-seq), a high-throughput NextGen sequencing method for harnessing high-copy transposons. We illustrate the efficacy of Mu-seq by applying it to the Robertson's Mutator system in a large population of maize plants. A single Mu-seq library, for example, constructed from 576 different families (2304 plants), enabled 4, 723 novel, germinal, transposon insertions to be detected, identified, and mapped with single base-pair resolution. In addition to the specificity, efficiency, and reproducibility of Mu-seq, a key feature of this method is its adjustable scale that can accommodate simultaneous profiling of transposons in thousands of individuals. We also describe a Mu-seq bioinformatics framework tailored to high-throughput, genome-wide, and population-wide analysis of transposon insertions.

SUBMITTER: McCarty DR

PROVIDER: S-EPMC3806735 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Publications

Mu-seq: sequence-based mapping and identification of transposon induced mutations.

McCarty Donald R DR Latshaw Sue S Wu Shan S Suzuki Masaharu M Hunter Charles T CT Avigne Wayne T WT Koch Karen E KE

PloS one 20131023 10

Mutations tagged by transposon insertions can be readily mapped and identified in organisms with sequenced genomes. Collections of such mutants allow a systematic analysis of gene function, and can be sequence-indexed to build invaluable resources. Here we present Mu-seq (Mutant-seq), a high-throughput NextGen sequencing method for harnessing high-copy transposons. We illustrate the efficacy of Mu-seq by applying it to the Robertson's Mutator system in a large population of maize plants. A singl ...[more]

PMID: 24194867

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Project description:In pursuing our long-term goals of identifying causal genes for mutant phenotypes in maize, we have developed a new, phenotype-to-genotype approach for transposon-based resources, and used this to identify candidate genes that co-segregate with visible kernel mutants. The strategy incorporates a redesigned Mu-seq protocol (sequence-based, transposon mapping) for high-throughput identification of individual plants carrying Mu insertions. Forward-genetic Mu-seq also involves a genetic pipeline for generating families that segregate for mutants of interest, and grid designs for concurrent analysis of genotypes in multiple families. Critically, this approach not only eliminates gene-specific PCR genotyping, but also profiles all Mu-insertions in hundreds of individuals simultaneously. Here, we employ this scalable approach to study 12 families that showed Mendelian segregation of visible seed mutants. These families were analyzed in parallel, and 7 showed clear co-segregation between the selected phenotype and a Mu insertion in a specific gene. Results were confirmed by PCR. Mutant genes that associated with kernel phenotypes include those encoding: a new allele of Whirly1 (a transcription factor with high affinity for organellar and single-stranded DNA), a predicted splicing factor with a KH domain, a small protein with unknown function, a putative mitochondrial transcription-termination factor, and three proteins with pentatricopeptide repeat domains (predicted mitochondrial). Identification of such associations allows mutants to be prioritized for subsequent research based on their functional annotations. Forward-genetic Mu-seq also allows a systematic dissection of mutant classes with similar phenotypes. In the present work, a high proportion of kernel phenotypes were associated with mutations affecting organellar gene transcription and processing, highlighting the importance and non-redundance of genes controlling these aspects of seed development.

Dataset Information

Mu-seq: sequence-based mapping and identification of transposon induced mutations.

Publications

Mu-seq: sequence-based mapping and identification of transposon induced mutations.

Similar Datasets

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